| BackgroundAn increasing number of studies have shown that polymorphisms in the human genome are closely associated with the occurrence and progression of tumors.Different genotypes of the same gene may play completely opposite roles in different tumor tissues.As an important tumor-inhibiting factor,P53 regulates the cell cycle and induces cell apoptosis and cell differentiation.Therefore,polymorphisms in P53 likely affect the progression and prognosis of prostate cancer.MDSCs play a pivotal role in cancer progression by suppressing immune response.The increasing frequency and phenotype of circulating MDSCs in peripheral blood have been reported in many types of cancers,both in the preclinical models and human patients.Several reports also have shown increased infiltration of MDSCs in cancers including breast cancer,lung cancer,and multiple myeloma,both in the primary tumor and metastatic sites.However,due to their heterogeneity,the frequency,phenotype and suppressive function in patients with cancer are highly debated and it is still not clear whether one subset is predominant over the other,especially in prostate cancer.ObjectivesThis paper mainly treats two factors which are potentially clinically significant and respectively important for the development and progression of prostate cancer,P53 gene and and myeloid-derived suppressive cells(MDSCs)as a starting point to study the impacts of these two factors on prostate cancer.It is convinced that our results would enrich our understanding of prostate cancer,its developmental mechanism,and provide valuable reference and a cue for the molecular target sites or provide a theoretical basis for the treatment of prostate cancer from many anglesMethodCommercialized two human LNCaP and PC3 prostate cancer cells cultured in the laboratory under a conventional cell culture system,.I.e.,containing 10%fetal bovine serum in RPMI 1640 culture medium.To transfect expressional plasmids,we used Lipofectamine 2000.Followed by conventional processes we used half-dry transfer Western Blot to detect the cell transfection efficiency.In order to detect the proliferation of different transfected cells we performed MTT colorimetric assay,followed by PI staining combined with flow cytometry for cell cycle analysis and Annexin V and PI staining combined with flow cytometry to detect cell apoptosis.In order to identify the relationship between MDSCs with prostate cancer,we first take the peripheral blood mononuclear cells from disease group and the control group and went through cell separation and following flow cytometry analysis.In flow cytometry analysis,the PBMC were marked with specific antibodies,including CD33-PE,CDllb-FITC,CD14-PerCP-Cy5.5,HLA-DR-APC and CD15-PE-Cy7.Then we conducted a multi-color flow cytometry through the system(FACSCanto Ⅱflow cytometry,BD Bioscience company).The data was analyzed with the software FlowJo.The percentage of MDSCs(CD33 + CD11b + HLA-DR-CD14-)in total live PBMC cells was calculated.After the serum of each group were isolated,ELISA assay was used to analyze the cytokines levels.Finally,we also studied the influence of MDSCs on T cell function.StatisticsThe experimental results was analyzed with SPSS statistical software and the average(Mean)±standard deviation(SD)was shown in results.Comparison between the two groups was analyzed with non-paired sample Student t-test.But the ANOVA or Mann-Whitney U test was used to determine statistically significant differences between the various groups.Spearman’s correlation analysis was used to analyze the correlation between MDSCs and cytokines.Survival curves were plotted using the Log-rank test and comparison method according KaplanMeier.This study set P value less than 0.05 as statistically significant difference.ResultsIt showed that wild P53(V/V)and mutant P53(G/G)overexpression can inhibit cell proliferation of two kinds of commercially available prostate cancer cell lines PC3 and LNCaP,and this effect may be because wild P53(V/V)and mutant P53(G/G)overexpression can make two kinds of commercially available prostate cancer cell line PC3 and LNCaP cells to arrest in GO/G1 phase and start a apoptosis.In addition,in these three aspects,the effect of mutant type P53 is more obvious when compared with the wild type P53.Therefore,we believe that P53 polymorphism may affect cancer development,particularly in the prostate cancer.The results show that there is a significant increase inCD33+CDllb+HLA-DR-CD14-marked MDSCs cells of blood circulation in patients with prostate cancer.Bycomparing blood samples from normal individuals,patients with BPH individuals and various stages of cancer,we identified a significant increase in the proportion of these MDSCs in the blood with the developmental stage of the cancer,and this phenomenon further correlates with the serum IL-8/IL-6 levels.Abnormal accumulation of MDSCs is an important mechanism for deficient T cell responses in cancer patients.These data not only enrich our understanding of immunobiology of prostate cancer,also may have some importance in the promotion of research to develop MDSCs suppression method and IL-8/IL-6 directed therapy.Conclusion1.Wild P53(V/V)and mutant P53(G/G)overexpression can inhibit proliferation of prostate cancer cell lines PC3 and LNCaP cells of two commercially available,and the effect of mutant compared with wild-type effect is obvious.2.Wild P53(V/V)and mutant P53(G/G)overexpression can make two kinds of commercially available prostate cancer cell line PC3 and LNCaP cells to increasingly arrest at G0/G1 phase.And similarly,and the effect of mutant compared with wild-type effect is obvious.3.Wild P53(V/V)and mutant P53(G/G)overexpression can cause increased apoptosis in two commercial prostate cancer cell line PC3 and LNCaP cells.And similarly,and the effect of mutant compared with wild-type effect is obvious.4.MDSCs ratio significantly increased with the development stage of the cancer.It also has a significant correlation with the amount of serum IL-8/IL-6.6.Tumor-derived MDSC having immunosuppressive effects in vitro can lead to abnormal T-cell activation. |
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